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Human T-lymphocytes activated by phytohemagglutin acquire insulin receptors in culture. Saturation analysis of insulin-binding activity in the presence of competing ligand revealed curvilinear Scatchard plots. Insulin receptors were not regulated by insulin before mitogen activation and culture of T-lymphocytes. However, insulin-induced downregulation of insulin receptors was: (1) demonstrable in receptor-positive cells, (2) dependent on insulin concentration, (3) temporally unrelated to insulin internalization, and (4) prevented by culture at 4 degrees C but not by cycloheximide at 37 degrees C. Recovery of insulin receptors required further culture of cells in media depleted of insulin for 24 h. Scatchard analysis revealed loss of receptor number without changes in receptor affinity. Insulin-induced increases in glucose transport and oxidation were demonstrable in receptor-positive cells but not in receptor-negative cells. However, these effects were extremely time-dependent. After a 2-h exposure of cells to 10(-8) M insulin, increases in glucose transport were no longer demonstrable. Elution of bound insulin from these cells followed by re-exposure to insulin depressed glucose transport in them. Recovery from this hyporesponsive, desensitized state required a 6-h culture in insulin-depleted media. Glucose oxidation of desensitized cells could be stimulated by spermine but not by insulin. These studies demonstrate the activated human T-lymphocyte is an insulin-sensitive tissue that is capable of limiting its physiologic response to insulin by receptor- and postreceptor-mediated mechanisms.

Octyl beta-glucoside (1%), a dialyzable detergent, was used to solubilize the insulin receptor of the turkey erythrocyte membrane. Insulin binding capacity was stable for at least 1 week when the receptor was kept in 1% octyl beta-glucoside at 4 degrees C. The binding properties of the solubilized receptor were examined at detergent concentrations above (1%) and below (0.6%) the critical micelle concentration. A reduction in insulin binding occurred when the detergent concentration was raised above the critical micelle concentration, due to an apparent decrease in the number of binding sites. The specificity of the receptor for insulin analogues was preserved, and the relative affinity of the solubilized receptor, desoctapeptide insulin greater than proinsulin greater than porcine insulin, was similar in 0.6% and 1% detergent. Addition of divalent cations increased insulin binding to a similar extent at both detergent concentrations, but there was a slightly greater stimulation of binding in 0.6% detergent as compared to 1% detergent. The pH optimum for binding was not affected by changes in the detergent concentration. These results indicate that the insulin receptor can be successfully solubilized by octyl beta-glucoside and that the binding activity is quite stable. Therefore, octyl beta-glucoside may be a useful detergent for purification of this receptor. In addition, the data indicate that the binding properties of the insulin receptor can be affected by changes in the physical state of the octyl beta-glucoside.

Previous studies using inhibitors have suggested that protein synthesis is necessary for "down-regulation" of insulin receptors. We have tested this hypothesis without the use of inhibitors by studying the ability of cells of the erythroid series to down-regulate their insulin receptors in vitro. The cells tested include mature erythrocytes and reticulocytes from rabbits and Friend erythroleukemia cells (a model for the basophilic erythroblast, a primitive nucleated erythrocyte). All cells were maintained at 37 degrees C for 18 hr +/- insulin (10(-8)M). Cultures were then incubated with phosphate buffered salines (pH 7.0) at 30 degrees C for 40 min to remove bound insulin. Receptors were quantitated by computerized analysis of Scatchard plots of subsequent insulin binding studies. Cells fully capable of both mRNA synthesis and protein synthesis, such as the undifferentiated and differentiated Friend erythroleukemia cell, had reduction of insulin receptors at 60% and 43%, respectively. Reticulocytes, which were capable of protein synthesis but not mRNA synthesis, had decreases of 25%-30% in 8 separate experiments. Mature erythrocytes, capable of neither RNA nor protein synthesis had no significant changes in receptor concentrations. Since mature erythrocytes do not "down-regulate" their insulin receptor concentration, studies of these receptors in erythrocytes of patients should be interpreted with caution.

The detergent-solubilized avian insulin receptor retains negative cooperativity and other binding properties of the membrane bound form. On gel filtration the receptor elutes as a single peak with a Stokes radius of 72 A. Preincubation of the receptor with low levels of insulin leads to the formation of a second, smaller form with a Stokes radius of 40 A. The percent of receptor in this second peak is proportional to the insulin concentration and correlates well with the insulin-induced increase in dissociation rate (negative cooperativity). Both the isolated high molecular weight and the isolated low-molecular-weight forms of the receptor re-equilibrate in the presence of insulin and, upon refiltration of either isolated peak, both forms of the receptor are obtained. These results are compatible with a model of the insulin receptor in which a tetrameric form can dissociate to a monomeric form as a concomitant of negative cooperativity.

The Friend erythroleukemia cell has an insulin receptor with all the properties of mammalian insulin receptors: rapid, reversible, and saturable binding of insulin; specific for insulin and insulin analogs; inversely proportional to temperatures; sharply pH dependent (optimum = 8.0); and demonstrated ligand-induced accelerated dissociation consistent with negative cooperativity. There were 17,200 sites per cell. After induction by dimethylsulfoxide, 80% of the cells became benzidine positive (i.e., contained hemoglobin). The receptor concentration dropped to 4300 sites per cell, while the remaining receptors retained all the initial binding characteristics. This loss of receptors could not be attributed directly to either dimethylsulfoxide or changes in cell size. Thus, during the process of differentiation, the concentration of insulin receptors in the Friend erythroleukemia cell decreases.

The insulin receptor was solubilized from turkey erythrocyte membranes by extraction with 1% beta-octylglucopyranoside. Insulin binding was enhanced when the solubilized material was reconstituted in phospholipid vesicles. The affinity of the reconstituted vesicles for various insulins was similar to that of the intact membranes: porcine insulin greater than proinsulin greater than desoctapeptide insulin. A curvilinear Scatchard plot was obtained for insulin binding to the reconstituted system at 15 degrees C. A high affinity association constant of 1.4 x 10(9) M-1 was obtained from the Scatchard plot. This is a four-fold increase over the value for the turkey erythrocyte membrane, which contains more highly saturated phospholipids. This suggests that the insulin receptor may be sensitive to the lipid composition of the membranes in which it is embedded.

Treatment of phytohemagglutinin (PHA) activated human T lymphocytes with tunicamycin, an antibiotic that specifically inhibits asparagine-linked N-glycosylation of proteins, totally blocked the normal emergence of insulin receptors on these lymphocytes and their cellular proliferation during culture in a dose-dependent manner. Carbohydrate incorporation into protein was inhibited 82% by 0.5 microgram/mL while leucine incorporation was unaffected. Tunicamycin exposure of activated T lymphocytes, which had acquired insulin receptors during culture, reduced cellular insulin binding by 35% to 84% and reduced PHA binding to 40% of control levels within 24 hours. Scatchard analysis revealed decreases in insulin binding capacity but not affinity. Similar treatment with cycloheximide only decreased insulin binding by 12%. These findings suggest N-glycosylation of proteins is a necessary biochemical event (1) for the emergence and maintenance of insulin receptors on mitogen activated T lymphocytes, and (2) for mitogen activated T lymphocytes to undergo cell division.

Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor. The solubilized insulin receptor has a Stokes radius of 70 A and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin. On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25--30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5. These data suggest that the insulin receptor retains its properties in the absence of the lipid bilayer of the membrane. Complete purification will be difficult due to a lack of stability under a number of conditions.

We have altered the phospholipid composition of the plasma membranes of Ehrlich ascites cells, grown in mice and studied the effects on the properties of the insulin receptor of this cell. The insulin receptor of the Ehrlich cell demonstrated all of the binding characteristics of mammalian insulin receptors: specificity for insulin and insulin analogs, saturability, inverse relationship of steady-state binding levels to temperature, and negative cooperativity. Cellular phospholipids enriched in monounsaturated fatty acyl groups were produced by growth in animals that were maintained on a diet rich in coconut oil; cellular phospholipids enriched in polyunsaturated fatty acyl groups were produced in animals fed sunflower oil. Insulin receptors were present in the normal cells at 180,000 sites/cell but this fell to 125000 (P less than 0.001) in cells enriched in monounsaturated fatty acids and rose to 386,000 (P less than 0.001) in cells enriched in polyunsaturated fatty acids. The normal cells had affinity constants (Ke and Kf) of 0.03 and 0.01 nM-1. The cells enriched in monounsaturated fatty acids had an increase in these affinity constants to 0.06 and 0.03 nM-1 whereas values of 0.01 and 0.005 mM-1 were obtained in the cells enriched in polyunsaturated fatty acids (all comparison P less than 0.001). Thus, increased unsaturation of plasma membrane phospholipids, produced by dietary manipulations, was associated with an increase in insulin receptor number but a decrease in binding affinity. In contrast, increased saturation of the phospholipids of the plasma membrane was associated with a decrease in receptor number and an increase in affinity. The results can be explained by a model in which the insulin receptor is assumed to be multimeric.

Primary human T lymphocytes that have been mitogen activated in chemically defined medium express cell surface insulin receptors. The receptor is identical to other mammalian insulin receptors in binding properties, including: pH dependency, ligand affinity, hormone specificity, and cooperative interactions. Scatchard plots are curvilinear and a ligand-induced increase in dissociation, the property normally associated with "negative cooperativity", is kinetically demonstrable. In vitro insulin treatment of the receptor-negative, resting T lymphocyte slightly enhances the degree of insulin binding which emerges following cellular activation. Insulin treatment of receptor-positive lymphoblasts results in insulin receptor "down-regulation". These findings indicate that T lymphoblast insulin receptor concentrations are not significantly influenced by insulin before their emergence but are dramatically regulated by insulin following their appearance at the cell surface.

The turkey erythrocyte membrane insulin receptor was solubilized and reconstituted into vesicles composed of either soy or dimyristoyl phosphatidylcholine. Reconstitution with soy phosphatidylcholine provided a lipid environment containing 43% unsaturated fatty acids, as compared with 82% saturated fatty acids in the dimyristoyl phosphatidylcholine preparation. After reconstitution, both species of vesicles were isolated from a 2 to 30% continuous sucrose gradient at a density of 1.071 g/ml. Scatchard analysis of binding data obtained at 15 degrees C revealed that the reconstituted receptor had a greater affinity for [125I]iodoinsulin in the saturated lipid environment (Ke = 0.167 nM-1; K1 = 2.18 nm-1) than in the unsaturated lipid environment (Ke = 0.0162 nM-1; K1 = 0.479 nm-1). Low affinity binding also was increased in the saturated vesicles. These increases were paralleled by a reduction in the number of available insulin binding sites in the saturated lipid environment. There was no difference, however, in the relative affinity of the reconstituted receptor preparations for insulin or proinsulin. Electron microscopy and gel filtration indicated that the binding differences are not due to differences in vesicle size. They also are not due to differences in the orientation of the receptor within the lipid bilayer, for its sensitivity to trypsin digestion was similar in both types of vesicles. Solubilization studies with 1% beta-octylglucoside indicated, however, that the dimyristoyl phosphatidylcholine vesicles incorporated a slightly lesser amount of insulin receptor. Similar results were also observed at 37 degrees C. These results suggest that the membrane lipid environment, especially the degree of unsaturation of the phospholipid fatty acyl chains, can influence the binding properties of the insulin receptor.

Cultured human Y79 retinoblastoma cells bind [125I]iodoinsulin in a manner similar to that of other CNS and peripheral tissues. The only difference noted between the insulin binding properties of the Y79 cells and other CNS preparations is that insulin binding to Y79 cells is down-regulated by prolonged exposure of the cells to insulin. By contrast, studies with the various brain preparations indicate that the brain insulin receptor is not down-regulated by circulating levels of insulin. Insulin binding to Y79 cells exhibits negative cooperativity, has a pH optimum of 7.8, is responsive to cations, and gives a curvilinear Scatchard plot. Y79 cell insulin binding capacity is 26 fmol/100 micrograms of cell protein, corresponding to about 125,000 binding sites per cell. These findings are the first to report insulin binding in a human cell line of retinal origin. The characterization of the insulin binding in this cell line may facilitate an understanding of the relationship between insulin and its specific functions in the human retina.

Avian erythrocytes possess insulin receptors which have binding properties that are virtually identical to those of the well studied mammalian insulin receptors. The affinity for porcine insulin was identical for the turkey and mammalian receptors over the entire range of insulin concentrations, as was the affinity of each of four insulin analogues which differed 300-fold in biological potency. Insulin induced acceleration of dissociation (i.e., the negatively cooperativite site-site interaction) was indistinguishable over a 10(6) range of insulin concentrations. Sharp pH dependence of binding was identical for turkey and mammalian receptors. The effects of temperature on association, dissociation and steady state binding were also identical. Thus, although birds and mammals have evolved separately for 300 million years there has been little change in the properties of the insulin receptor over this time period.

The erythrocyte (RBC) has received recent interest as a cell model to examine insulin receptor status in humans. In the present study we have compared the insulin receptors on mature RBCs and monocytes of four hyperinsulinemic obese patients in the fed state and after a 14-day fast (less than 50 cal/day). Insulin binding in the basal (fed) state was described in RBC and monocytes due predominantly to a decrease in the receptor concentration in both cell types. After a 14-day fast, insulin binding to both RBCs and monocytes increased significantly in each patient. Maximal binding of [125I]iodoinsulin to RBCs increased by 29% (range, 20-46%), and binding to monocytes increased by 116% (range, 46-321%). In response to the fast, the concentration of insulin needed to inhibit binding by 50% decreased from 5 to 2 ng/ml in RBC and from 3 to 1 ng/ml in monocytes. Conventional and computer-fitted Scatchard analyses demonstrated no change in the receptor concentration of RBCs of any patient, whereas the receptor concentration of monocytes increased by more than 50% in two of the four patients and by 40% for the group. Thus, in response to the fast, the direction of the change in insulin binding was similar in the RBCs and monocytes, whereas the magnitude and, in certain patients, the mechanism of the binding increase differed.